Abstract
Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of a nuclear receptor superfamily and acts as a ligand-dependent transcription factor, playing key roles in maintenance of adipose tissue and in regulation of glucose and lipid homeostasis. This receptor is the target of thiazolidinediones, a class of antidiabetic drugs, which improve insulin sensitization and regulate glycemia in type 2 diabetes. Despite the beneficial effects of drugs, such as rosiglitazone and pioglitazone, their use is associated with several side effects, including weight gain, heart failure, and liver disease, since these drugs induce full activation of the receptor. By contrast, a promising activation-independent mechanism that involves the inhibition of cyclin-dependent kinase 5 (CDK5)-mediated PPARγ phosphorylation has been related to the insulin-sensitizing effects induced by these drugs. Thus, we aimed to identify novel PPARγ ligands that do not possess agonist properties by conducting a mini-trial with 80 compounds using the sequential steps of thermal shift assay, 8-anilino-1-naphthalenesulfonic acid fluorescence quenching, and a cell-based transactivation assay. We identified two non-agonist PPARγ ligands, AM-879 and P11, and one partial-agonist, R32. Using fluorescence anisotropy, we show that AM-879 does not dissociate the NCOR corepressor in vitro, and it has only a small effect on TRAP coactivator recruitment. In cells, AM-879 could not induce adipocyte differentiation or positively regulate the expression of genes associated with adipogenesis. In addition, AM-879 inhibited CDK5-mediated phosphorylation of PPARγ in vitro. Taken together, these findings supported an interaction between AM-879 and PPARγ; this interaction was identified by the analysis of the crystal structure of the PPARγ:AM-879 complex and evidenced by AM-879’s mechanism of action as a putative PPARγ non-agonist with antidiabetic properties. Moreover, we present an optimized assay pipeline capable of detecting ligands that physically bind to PPARγ but do not cause its activation as a new strategy to identify ligands for this nuclear receptor.
Highlights
Obesity and type 2 diabetes are characteristic pathologies of the metabolic syndrome and have reached epidemic proportions in recent years [1]
Ligand-bound peroxisome proliferator activator receptor γ (PPARγ) presents a higher structural stability than the unbound receptor [37, 38]; the first step in our ligand screening involved searching for molecules that increased the stability of PPARγ using a Thermal Shift Assay (TSA)
We developed a new approach to screen for PPARγ ligands following a pipeline of assays that consider the biophysical characteristics of this nuclear receptor, aiming to identify ligands that bind to the receptor but do not promote coactivator recruitment, phosphorylation of S273, and adipogenic activity
Summary
Obesity and type 2 diabetes are characteristic pathologies of the metabolic syndrome and have reached epidemic proportions in recent years [1]. They are associated with an increased risk of cardiovascular disease and stroke. Recent studies have improved our knowledge about these conditions and have suggested that adipose tissue is at the center of the metabolic syndrome [3]. Other reports have shown that peroxisome proliferator-activated receptor gamma (PPARγ) plays key roles in adipose tissue, acting as a master regulator of fat cell biology [4,5,6]. The most clinically relevant finding in this field was the well-established link between PPARγ activity and insulin sensitivity [7]
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